In an effort to develop sensitive and precise probes with which to investigate the biochemical and biological consequences of carcinogen-induced genomic damage, scientists have elicited animal antibodies against carcinogen-DNA adducts. (Poirier, M. C. "The Use of Carcinogen-DNA Adduct Antisera for Quantitation and Localization of Genomic Damage in Animal Models and the Human Population", Environmental Mutagenesis 6:879-887 (1984)). The use of antibodies to detect chemical carcinogen-induced DNA damage involves quantitative determination using antisera specific for carcinogen-DNA adducts.
In recent years immunoassays for a variety of carcinogen-DNA adducts have been developed, many involving the use of monoclonal antibodies. (Wild, C. P., Smart G., Saffhill, R., and Boyle J. M. Radioimmunoassay of O.sup.6 -methyldeoxyguanosine in DNA of cells alkylated in vitro and in vivo. Carcinogenesis (Lond.), 4: 1605-1609, 1983; Hsu, I-C., Poirier, M. C. Yuspa, S. H. Grunberger, D., Weinstein, I. B., Yolken, R. H., and Harris, C. C. Measurement of benzo(a)pyrene-DNA adducts by enzyme immunoassay and radioimmunoassay. Cancer Res., 41: 1091-1095, 1981; Groopman, J. D., Haugen, A., Goodrich, G. R., Wogan, G. N., and Harris, C. C. Quantitation of aflatoxin B.sub.1 -modified DNA using monoclonal antibodies, Cancer Res., 42: 3120-3124, 1982; Poirier, M. C. The use of carcinogen-DNA adduct antisera for quantitation and localization of genomic damage in animal models and the human population. Environ. Mutagen., 6: 879-887, 1984). Preparation of monoclonal antibodies able to detect particular antigens and for the production by hybridoma technology of pure antibodies is well-known in the art. These assays are highly sensitive being able to detect as little as 0.5 .mu.mol adduct per mol deoxyguanosine. Carcinogen-DNA adducts have also been detected in humans with the aid of immunoassays. (Harris, C. C., Vahakangas, K., Newman, M. J., Tivers, G. E., Shamsuddin, A., Sinopoli, N., Mann, D. L., and Wright, W. E., Detection of benzo(a)pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers, Proc. Natl. Acad. Sci. USA, 82: 6672-6676, 1985; Umbenhauer, D., Wild, C. P., Montesano, R., Saffhill, R., Boyle, J. M., Huh, N., Kirstein, U., Thomale, J., Rajewsky, M. F., and Lu, S. H., O.sup.6 -Methyldeoxyguanosine in oesphageal DNA among individuals at high risk of oesophageal cancer. Int. J. Cancer, 36: 661-665, 1985.)
Notwithstanding the above, a problem with immunoassays is the production of a suitable antibody. If one is interested in detecting and measuring a specific DNA adduct, there is little point in eliciting antibodies against a different albeit related molecule. Furthermore, while the limits of sensitivity and specificity are theoretically high, in practice obtaining suitable antibodies is a rare event.
Indeed, although the technique underlying hybridoma technology is well recognized, nevertheless, the results obtained by its use clearly are unpredictable. Hybridoma technology is an empirical art in which the routinist is unable to foresee what particular antibodies will be produced and which specific surface antigens will be recognized by them. Only by actually carrying out the requisite steps can the nature of the monoclonal antibodies be determined and ascertained.
Recently the modified nucleosides 8R, 6R- and 8S, 6S-3-(2-deoxy-3-.beta.-erythro-pentofuranosyl) 5, 6, 7, 8-tetrahydro-8-hydroxy-6-methylpyrimido(1, 2-a)purine-10(3H)one were identified in DNA which had been exposed in vitro to crotonaldehyde (CH.sub.3 CH.dbd.CHCHO). The same modified deoxyguanosines may also form as a result of hydroxylation of N-nitrosopyrrolidine (NPYR). Both crotonaldehyde and NPYR are tumorigenic in rats with NPYR being the more potent of the two. Crotonaldehyde is also mutagenic toward Salmonella typhimurium and is commonly found in the human environment. NPYR is found in cooked bacon, mainstream and sidestream tobacco smoke, and as a trace contaminant of various foodstuffs.
In order to study the role of the cyclic DNA adducts in the mechanism of action of crotonaldehyde and NPYR a sensitive assay was heretofore needed to measure the formation of such adducts.